The long-term objective of this project is to better understand the molecular mechanisms that underlie retinal pigment epithelium (RPE)-specific gene expression. The RPE is important for the function and survival of retinal photoreceptors. Abnormalities in the RPE have been implicated in the pathogenesis of photoreceptor degeneration both in the RCS rat and in human age-related macular degeneration (AMD), the leading cause of irreversible blindness in elderly Americans. In addition, mutations in RPD65, RLBP1, TIMP3, and VMD2, four genes that are specifically or preferentially expressed in the RPE, have been shown to cause Leber's Congenital Amaurosis (LCA), retinitis pigmentosa (RP), Sorsby's fundus dystrophy (SFD), and vitelliform macular dystrophy (Best's disease), respectively. However, despite the importance of the RPD, and the importance of genes that are specifically or preferentially expressed in the RPE, little is known about the molecular mechanisms that regulate RPE-specific gene expression. Therefore, the goal of this proposal is to begin elucidating such mechanisms using human VMD2 (Best's disease gene) as a model system. More specifically, the following questions will be addressed: What are the cis-acting DNA elements that regulate VMD2 expression? Do different RPE-specific genes share common regulatory elements? What are the trans-acting factors that bind to the DNA regulatory elements? Are mutations in RPE promoters and/or RPE transcription factors involved in human disease? For answering these questions, DNA elements in the promoter region of VMD2 will be identified by sequence comparison with the other RPE-specific promoters and by biochemical analysis using DNase I footprint and electrophoretic mobility shift (EMSA) assays. The promoter region of VMD2 will be functionally analyzed using a combination of transient transfection and transgenic mouse approaches. Finally, to clone and characterize cDNAs for the trans-acting that bind to the DNA regulatory elements in the VMD2 promoter, we will use the yeast one-hybrid approach with a human RPE cDNA library. Increased understanding of RPE gene regulation should help provide new insights into diseases involving abnormalities of RPE gene expression. In addition, the knowledge will be useful in the development of RPE-targeted gene therapy and the generation of transgenic mice with targeted expression of exogenous proteins to the RPD. Furthermore, since Best's disease shares some clinical and histological features with AMD, studies of human VMD2 expression may have important implications for understanding macular degeneration.